In 2002, the Nobel Prize in chemistry was shared by Koichi Tanaka for the concept of “matrix assisted” laser desorption ionization (MALDI), which allowed large molecules to be analyzed intact using mass spectrometry. In this technique, the target particle (analyte) is coated by a matrix chemical, which preferentially absorbs light (often ultraviolet wavelengths) from a laser. In the absence of the matrix, the biological molecules would decompose by pyrolysis when exposed to a laser beam in a mass spectrometer. The matrix chemical also transfers charge to the vaporized molecules, creating ions that are then accelerated down a flight tube by the electric field (Brown, 1995; Knochenmuss, 1996). Microbiology and proteomics have become major application areas for mass spectrometry; examples include the identifaction of bacteria (Carbonnelle, 2010), discovering chemical structures, and deriving protein functions (Karas, 1987; Cotter, 1994, 1997). Danielewicz and co-workers (2011) report the use of MALDI mass spectrometry for lipid profiling of algae.
The coated analyte particles, which are often intact microbes are then analyzed using MALDI Time of Flight (TOF) mass spectrometry. FIG. 1 shows the “conventional” MALDI TOF mass spectrometry process. A liquid, usually comprised of an acid, such as tri-fluoro-acetic acid (TFA), and a MALDI matrix chemical such as alpha-cyano-4-hydroxycinnamic acid, is dissolved in a solvent and added to the analyte. Solvents include acetonitrile, water, ethanol, and acetone. TFA is normally added to suppress the influence of salt impurities on the mass spectrum of the analyte. Water enables hydrophilic proteins to dissolve, and acetonitrile enables the hydrophobic proteins to dissolve. The MALDI matrix solution is spotted on to the analyte on a MALDI plate 10 to yield a uniform homogenous layer of MALDI matrix material on the analyte. The solvents vaporize, leaving only the recrystallized matrix with the analyte spread through the matrix crystals. The acid partially degrades the cell membrane of the analyte making the proteins available for ionization and analysis in a TOF mass spectrometer. The coated plate is then analyzed in a TOF mass spectrometer.
Other MALDI matrix materials include 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid), α-cyano-4-hydroxycinnamic acid (α-cyano or α-matrix) and 2,5-dihydroxybenzoic acid (DHB) as described in U.S. Pat. No. 8,409,870.
The accuracy of MALDI mass spectrometry can be higher than that obtainable using traditional chemical techniques. Carbonelle et al. (2010) provides an excellent analysis of MALDI mass spectrometry bacterial identification in clinical microbiological laboratories, and conclude that the identification of bacteria, yeast, and fungi by MALDI mass spectrometry was the fastest technique for routine microbiology. Cherkaoui and co-workers (2010) compared mass spectrometry with conventional biochemical test system identifications for clinically relevant bacteria. Discordant results were resolved with “gold standard” 16S rRNA gene sequencing. The first MALDI mass spectrometry system (Bruker) gave high-confidence identifications for 680 isolates, of which 674 (99.1%) were correct; the second mass spectrometry system (Shimadzu) gave high-confidence identifications for 639 isolates, of which 635 (99.4%) were correct. FIG. 2 shows typical spectra for whole colonies of different species. It should be noted that the same species can yield differing spectra on account of the growing conditions, the growth phase, and the matrix used to obtain the spectra. However, microbial indentification is accurate, owing to the fact that many of the peaks associated with protein biomarkers are preserved.
Conventional MALDI mass spectrometry can be used to analyze cultured bacteria samples, but is generally not amenable to analysis of single organisms. Furthermore, if the sample contains a mixture of bacteria, the spectra generated would contain the characteristics of all of the species in the sample; hence, the spectra are cluttered and difficult to deconvolute. Both of these issues can be overcome by culturing the sample, which creates an abundance of the target bacteria in a colony. However, this approach has two drawbacks, namely, (1) it works only for culturable targets, and (2) because sample preparation occurs off-line in the lab, it incurs an associated labor cost and is time intensive. The conventional MALDI mass spectrometry is not amenable to real time analysis of bioaerosols in a sample, for example, biological contaminant particles in ambient air.
Aerosol Time of Flight Mass Spectrometry (ATOFMS) can be used to perform real-time measurements of sampled particles or aerosols. Measurements of individual particle composition and size made by ATOFMS provide valuable insights into the sources of these particles. ATOFMS has been used for the detection of aerosols and health effects studies. For example, TSI, Inc. (Shoreview, Minn.) has manufactured mass spectrometers to yield particle size and composition information for particles that range from 0.03 to 3 micrometers in size. The total inlet air flow rate to the spectrometer is 0.1 L/min. Ambient aersosol particle loadings are typically 10 to 1000 particles per mL for particles in the 0.03 to 3 micrometers size range. ATOFMS uses an aerodynamic sizing technique to sample and size particles based on each particle's transit time between two laser beams. The particles are then illuminated using a UV laser. The laser vaporizes and ionizes at least some of the molecules which comprise each illuminated particle, thus generating ions with specific mass to charge ratios (m/z). The electric field inside the mass spectrometer accelerates these charged particles and measures the time-of-flight to a detector to yield a unique fingerprint spectrum. This measured spectrum can be compared with database information to provide information on chemical composition. ATOFMS quickly identifies the specific chemical compounds that make up each particle. Hundreds or perhaps thousands of particles can be analyzed per second using this technique, providing real-time information on the nature of the aerosol.
Particles comprising proteins and other bio-organic molecules (bioaerosols) however tend to absorb the intense UV laser light and decompose by pyrolysis, making the ATOFMS analysis technique ineffective. In order to analyze bioaerosols, each of the particles in the aerosol must be coated with the MALDI matrix. MALDI matrix coating of analyte aerosol particles prior to introduction into the ATOFMS was first described by Stowers (2000), and then by van Wuijckhuijse (2004). The advantage of analyzing particles in a bioaerosol one particle at a time is that each particle is a representative of the “pure sample” of the constituent proteins and other high molecular weight molecules. In the case of a single airborne bacterium, it represents a “pure culture” of that one organism. The ambient aerosol (analyte) is first passed through a hot zone which contains a relative high vapor pressure MALDI matrix material. The matrix vapor mixes with the aerosol by diffusion and then enters into a cool zone. Here, the matrix condenses out of the vapor phase to form a matrix aerosol, some of which may coat the analyte aerosol particles. The analyte aerosol is dispersed within the matrix aerosol, but the mean particle diameter is considerably less than that of the matrix aerosol. The analyte aerosol particle typically has a particle size of between 0.5 to 15 micrometers. The analyte aerosol is now “vapor coated” with matrix. Mass spectrometry fingerprints obtained using the aerosol MALDI MS technique closely resemble those obtained from conventional MALDI mass spectrometry. For microbes, the fingerprint can be associated with specific proteins present in a particular organism. FIG. 3 compares measurements for single spores (diameter of about 1 micrometer) of Bacillus atrophaeus by van Wuijckhuijse (2010), with measurements from a conventional MALDI mass spectrometry (Hathout, 1999). As can be seen, key biomarkers are preserved across platforms. With conventional MALDI mass spectrometry, the fingerprint is obtained by co-averaging hundreds of spectra obtained from a single clump comprised of thousands of spores. In contrast, with aerosol MALDI mass spectrometry, the fingerprint was obtained by averaging 135 single-spore spectra.
The vapor coating technique of van Wuijckhuijse et al. has some notable shortcomings. First, vapor deposition of the conventional MALDI matrix chemical may not accurately simulate and replicate the homogeneous and uniform coating obtained during conventional liquid coating of the analyte (in FIG. 1). Further, as disclosed in U.S. Pat. No. 8,409,870, the technique required the use of a customized MALDI matrix material 2-mercapto-4,5-dialkylheteroarene and an alcohol. Suitable alcohols included methanol, ethanol and propanol. This is a significant limitation in that the conventional matrix chemicals, such as alpha-Cyano-4-hydroxycinnamic acid, 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid), and 2,5-dihydroxybenzoic acid (DHB) are prone to degrade during the vaporization step of the vapor deposition technique.
Therefore, a real-time and “on-the-fly” aerosol coater, capable of producing a coating of matrix material on the analyte particle, and preferably while using conventional matrix materials is desired.